A spectrofluorometric analysis to evaluate transcutaneous biodistribution of fluorescent nanoparticulate gel formulations

The investigation of the absorption of drug delivery systems, designed for the transport of therapeutic molecules inside the body, could be relatively simplified by the fluorophore association and tracking by means of bio-imaging techniques (i.e., optical in vivo imaging or confocal and multiphoton microscopy). However, when a fluorescence signal comes out from the skin, its specific detection can be problematic. Skin high autofluorescence can hinder the observation of administered exogenous fluorophores conjugated to drug delivery systems, making it more challenging to detect their biodistribution. In the present study, we have developed a method based on the spectrofluorometric analysis of skin samples to discriminate the fluorescent signal coming from administered fluorescent molecules from the background. Moreover, we gave a semi-quantitative evaluation of the signal intensity. Thus, we distinguished two gel formulations loading the fluorophore rhodamine B (called GEL RHO and GEL SLN-RHO). The two formulations of gels, one of which containing solid lipid nanoparticles (GEL RHO-SLN), were administered on skin explants incubated in a bioreactor, and the penetration was evaluated at different time points (2 and 6 hours). Cryostatic sections of skin samples were observed with confocal laser scanning microscopy, and a spectrofluorometric analysis was performed. Significantly higher signal intensity in the samples administered with SLN-RHO GEL, with a preferential accumulation in the hair bulbs, was found. Reaching also the deeper layers of the hair shaft after 6 hours, the solid lipid nanoparticles thickened with polymer represent a suitable drug delivery system for transcutaneous administration

Mitochondrial Features of Mouse Myoblasts Are Finely Tuned by Low Doses of Ozone: The Evidence In Vitro

The mild oxidative stress induced by low doses of gaseous ozone (O3) activates the antioxidant cell response through the nuclear factor erythroid 2-related factor 2 (Nrf2), thus inducing beneficial effects without cell damage. Mitochondria are sensitive to mild oxidative stress and represent a susceptible O3 target. In this in vitro study, we investigated the mitochondrial response to low O3 doses in the immortalized, non-tumoral muscle C2C12 cells; a multimodal approach including fluorescence microscopy, transmission electron microscopy and biochemistry was used. Results demonstrated that mitochondrial features are finely tuned by low O3 doses. The O3 concentration of 10 μg maintained normal levels of mitochondria-associated Nrf2, promoted the mitochondrial increase of size and cristae extension, reduced cellular reactive oxygen species (ROS) and prevented cell death. Conversely, in 20 μg O3-treated cells, where the association of Nrf2 with the mitochondria drastically dropped, mitochondria underwent more significant swelling, and ROS and cell death increased. This study, therefore, adds original evidence for the involvement of Nrf2 in the dose-dependent response to low O3 concentrations not only as an Antioxidant Response Elements (ARE) gene activator but also as a regulatory/protective factor of mitochondrial function

A Correlative Imaging Study of in vivo and ex vivo Biodistribution of Solid Lipid Nanoparticles

Purpose: Solid lipid nanoparticles are largely used in biomedical research and are characterized by high stability and biocompatibility and are also able to improve the stability of various loaded molecules. In vitro studies demonstrated that these nanoparticles are low cytotoxic, while in vivo studies proved their efficiency as nanocarriers for molecules characterized by a low bioavailability. However, to our knowledge, no data on the systemic biodistribution and organ accumulation of solid lipid nanoparticles in itself are presently available. Methods: In this view, we investigated the solid lipid nanoparticles biodistribution by a multimodal imaging approach correlating in vivo and ex vivo analyses. We loaded solid lipid nanoparticles with two different fluorophores (cardiogreen and rhodamine) to observe them with an optical imager in the whole organism and in the excised organs, and with fluorescence microscopy in tissue sections. Light and transmission electron microscopy analyses were also performed to evaluate possible structural modification or damage due to nanoparticle administration. Results: Solid lipid nanoparticles loaded with the two fluorochromes showed good optic characteristics and stable polydispersity. After in vivo administration, they were clearly detectable in the organism. Four hours after the injection, the fluorescent signal occurred in anatomical districts corresponding to the liver and this was confirmed by the ex vivo acquisitions of excised organs. Brightfield, fluorescence and transmission electron microscopy confirmed solid lipid nanoparticles accumulation in hepatocytes without structural damage. Conclusion: Our results support the systemic biocompatibility of solid lipid nanoparticles and demonstrate their detailed biodistribution from the whole organism to organs until the cells

In vivo and in vitro biodistribution of solid lipid nanoparticles

Solid lipid nanoparticles (SLN) are a versatile tool with a high potential of applications. They are constituted by biocompatible and biodegradable matrix with well-established safety profiles. Their matrix can improve the stability and bioavailability of labile molecules, assuring restrained release profile. Moreover, due to the physicochemical properties of lipids (i.e. the low melting temperature), SLN can be easily obtained by direct emulsification of the molten lipids and subsequent recrystallization, thus avoiding the use of potentially toxic solvents that are commonly required for the preparation of polymeric nanoparticles (1). SLN used for this study were produced by a protocol based on emulsification of the molten lipids in water by melt and ultra-sonication method (2), and were functionalized with polysorbate 80 (SLN-P80) for in vivo administration. In fact, it has been reported that this surfactant extend the nanoparticle circulation time in the blood, avoiding opsonization with the complement activation and uptake by the reticulo-endothelial system. The influence of nanoparticle composition and functionalization was investigated on morphology, dimension and inner structure by mean of cryogenic transmission electron microscopy (cryo-TEM), X-ray diffraction measurements, photon correlation spectroscopy (PCS) and sedimentation field flow fractionation (SdFFF) (2). SLN-P80 were labeled with cardiogreen fluorophore to allow their visualization in living organisms, and the in vivo biodistribution was evaluated with Fluorescent Luminescent Imaging (FLI) after intraperitoneal (i.p.) administration in a mice model. After the in vivo acquisition, mice were perfused and the excised organs (liver, kidney, spleen, brain, lung) were acquired to confirm SLN-P80 accumulation in specific districts (3). Organ tissues were also analyzed at light microscopy (LM) to evaluate SLN-P80 localization. After 4 h from systemic administration, SLN-P80 specifically accumulated in liver (Fig. 1a). LM revealed that no histological alteration is induced by SLN-P80 administration; however, a marked increase in lipid content (red oil staining) was found in hepatocytes, especially close to the centrilobular venula (Fig. 1b,c). To clarify this phenomenon, we investigated in vitro the uptake and intracellular fate of SLN-P80 in a murine cell line. 3T3 cells were incubated with SLN-P80 for 1, 4 and 24h, and then processed for both LM (red oil staining) and transmission electron microscopy (TEM). SLN-P80 administration induced accumulation of lipid droplets already after 1 h (Fig. 2a,b). At TEM scarce SLN-P80 were observed inside the cells: they occurred free in the cytoplasm, often in close proximity to lipid droplets showing a peripheral electron dense material (Fig. 2c). This suggests that SLN-P80 enter the cells by fusion with the plasma membrane, undergo rapid degradation and their components migrate, probably due to chemical affinity, into the lipid deposits

3D renal model for surgical planning of partial nephrectomy: A way to improve surgical outcomes

to evaluate the impact of 3D model for a comprehensive assessment of surgical planning and quality of partial nephrectomy (PN)

Impact of Rapid Urbanization on the Rates of Infection by Vibrio cholerae O1 and Enterotoxigenic Escherichia coli in Dhaka, Bangladesh

Bangladesh is a country where acute dehydrating diarrhea or cholera is common and is seen at least two times every year and additionally in natural disasters. In addition cholera cases have increased in the country, especially in urban settings such as in the capital city, Dhaka, where the number of hospitalized patients with more severe disease has tremendously increased. In the present observation, we have concentrated on determining the occurrence of diarrhoea caused by the two most common bacterial agents V. cholerae O1 and enterotoxigenic Escherichia coli (ETEC) in a densely populated, disease prone area Mirpur in Dhaka for two years from March 2008 to February 2010. Stool or rectal specimens from diarrheal patients coming to the ICDDR,B hospital from Mirpur were tested for the two bacterial pathogens. We found that V. cholerae O1 was the major bacterial pathogen and a cause of severe cholera disease in 23% of patients (2,647 of a total of 11,395 patients) from Mirpur. We surmise that cholera vaccines, as well as other public health tools that can target such high risk groups in the country, will be able to reduce the disease morbidity and the transmission of pathogens to improve the quality of life in urban settings

Interrupting the nitrosative stress fuels tumor-specific cytotoxic T lymphocytes in pancreatic cancer

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest tumors owing to its robust desmoplasia, low immunogenicity, and recruitment of cancer-conditioned, immunoregulatory myeloid cells. These features strongly limit the success of immunotherapy as a single agent, thereby suggesting the need for the development of a multitargeted approach. The goal is to foster T lymphocyte infiltration within the tumor landscape and neutralize cancer-triggered immune suppression, to enhance the therapeutic effectiveness of immune-based treatments, such as anticancer adoptive cell therapy (ACT). METHODS: We examined the contribution of immunosuppressive myeloid cells expressing arginase 1 and nitric oxide synthase 2 in building up a reactive nitrogen species (RNS)-dependent chemical barrier and shaping the PDAC immune landscape. We examined the impact of pharmacological RNS interference on overcoming the recruitment and immunosuppressive activity of tumor-expanded myeloid cells, which render pancreatic cancers resistant to immunotherapy. RESULTS: PDAC progression is marked by a stepwise infiltration of myeloid cells, which enforces a highly immunosuppressive microenvironment through the uncontrolled metabolism of L-arginine by arginase 1 and inducible nitric oxide synthase activity, resulting in the production of large amounts of reactive oxygen and nitrogen species. The extensive accumulation of myeloid suppressing cells and nitrated tyrosines (nitrotyrosine, N-Ty) establishes an RNS-dependent chemical barrier that impairs tumor infiltration by T lymphocytes and restricts the efficacy of adoptive immunotherapy. A pharmacological treatment with AT38 ([3-(aminocarbonyl)furoxan-4-yl]methyl salicylate) reprograms the tumor microenvironment from protumoral to antitumoral, which supports T lymphocyte entrance within the tumor core and aids the efficacy of ACT with telomerase-specific cytotoxic T lymphocytes. CONCLUSIONS: Tumor microenvironment reprogramming by ablating aberrant RNS production bypasses the current limits of immunotherapy in PDAC by overcoming immune resistance

"Delirium Day": A nationwide point prevalence study of delirium in older hospitalized patients using an easy standardized diagnostic tool

Background: To date, delirium prevalence in adult acute hospital populations has been estimated generally from pooled findings of single-center studies and/or among specific patient populations. Furthermore, the number of participants in these studies has not exceeded a few hundred. To overcome these limitations, we have determined, in a multicenter study, the prevalence of delirium over a single day among a large population of patients admitted to acute and rehabilitation hospital wards in Italy. Methods: This is a point prevalence study (called "Delirium Day") including 1867 older patients (aged 65 years or more) across 108 acute and 12 rehabilitation wards in Italian hospitals. Delirium was assessed on the same day in all patients using the 4AT, a validated and briefly administered tool which does not require training. We also collected data regarding motoric subtypes of delirium, functional and nutritional status, dementia, comorbidity, medications, feeding tubes, peripheral venous and urinary catheters, and physical restraints. Results: The mean sample age was 82.0 ± 7.5 years (58 % female). Overall, 429 patients (22.9 %) had delirium. Hypoactive was the commonest subtype (132/344 patients, 38.5 %), followed by mixed, hyperactive, and nonmotoric delirium. The prevalence was highest in Neurology (28.5 %) and Geriatrics (24.7 %), lowest in Rehabilitation (14.0 %), and intermediate in Orthopedic (20.6 %) and Internal Medicine wards (21.4 %). In a multivariable logistic regression, age (odds ratio [OR] 1.03, 95 % confidence interval [CI] 1.01-1.05), Activities of Daily Living dependence (OR 1.19, 95 % CI 1.12-1.27), dementia (OR 3.25, 95 % CI 2.41-4.38), malnutrition (OR 2.01, 95 % CI 1.29-3.14), and use of antipsychotics (OR 2.03, 95 % CI 1.45-2.82), feeding tubes (OR 2.51, 95 % CI 1.11-5.66), peripheral venous catheters (OR 1.41, 95 % CI 1.06-1.87), urinary catheters (OR 1.73, 95 % CI 1.30-2.29), and physical restraints (OR 1.84, 95 % CI 1.40-2.40) were associated with delirium. Admission to Neurology wards was also associated with delirium (OR 2.00, 95 % CI 1.29-3.14), while admission to other settings was not. Conclusions: Delirium occurred in more than one out of five patients in acute and rehabilitation hospital wards. Prevalence was highest in Neurology and lowest in Rehabilitation divisions. The "Delirium Day" project might become a useful method to assess delirium across hospital settings and a benchmarking platform for future surveys

Multiancestry analysis of the HLA locus in Alzheimer’s and Parkinson’s diseases uncovers a shared adaptive immune response mediated by HLA-DRB1*04 subtypes

Across multiancestry groups, we analyzed Human Leukocyte Antigen (HLA) associations in over 176,000 individuals with Parkinson’s disease (PD) and Alzheimer’s disease (AD) versus controls. We demonstrate that the two diseases share the same protective association at the HLA locus. HLA-specific fine-mapping showed that hierarchical protective effects of HLA-DRB1*04 subtypes best accounted for the association, strongest with HLA-DRB1*04:04 and HLA-DRB1*04:07, and intermediary with HLA-DRB1*04:01 and HLA-DRB1*04:03. The same signal was associated with decreased neurofibrillary tangles in postmortem brains and was associated with reduced tau levels in cerebrospinal fluid and to a lower extent with increased Aβ42. Protective HLA-DRB1*04 subtypes strongly bound the aggregation-prone tau PHF6 sequence, however only when acetylated at a lysine (K311), a common posttranslational modification central to tau aggregation. An HLA-DRB1*04-mediated adaptive immune response decreases PD and AD risks, potentially by acting against tau, offering the possibility of therapeutic avenues